1. Field of the Invention
The present invention relates to the identification of new muteins of fibroblast growth factor 21 that have reduced deamidation when compared to wild type fibroblast growth factor 21.
2. Description of the Related Art
Fibroblast growth factors are polypeptides widely expressed in developing and adult tissues (Baird et al., Cancer Cells, 3:239-243, 1991) and play crucial roles in multiple physiological functions including angiogenesis, mitogenesis, pattern formation, cellular differentiation, metabolic regulation and repair of tissue injury (McKeehan et al., Prog. Nucleic Acid Res. Mol. Biol. 59:135-176, 1998). According to the published literature, the FGF family now consists of at least twenty-three members, FGF-1 to FGF-23 (Reuss et al., Cell Tissue Res. 313:139-157 (2003).
Fibroblast growth factor-21 (FGF-21) has been reported to be preferentially expressed in the liver (Nishimura et al., Biochimica et Biophysica Acta, 1492:203-206, 2000); WO01/36640; and WO01/18172) and described as a treatment for ischemic vascular disease, wound healing, and diseases associated with loss of pulmonary, bronchia or alveolar cell function and numerous other disorders. More recently, FGF-21 has been shown to stimulate glucose-uptake in mouse 3T3-L1 adipocytes after treatment in the presence and absence of insulin, and to decrease fed and fasting blood glucose, triglycerides, and glucagon levels in ob/ob and db/db mice and 8 week old ZDF rats in a dose-dependant manner, thus, providing the basis for the use of FGF-21 as a therapy for treating diabetes and obesity (WO03/011213).
The development of recombinant DNA technology has made possible the production of foreign products such as muteins of FGF-21 in host cells in which exogenous DNA sequences coding for those products have been introduced. The advantage of this technology is that products can be produced in high yields, in highly purified form, with low risk of contamination such as viral contamination. These recombinant techniques have been widely used for the production of recombinant proteins in prokaryotic as well as eukaryotic host cells.
However, the large-scale production of recombinant products by these techniques is still limited, due to problems of expression efficiency of these exogenous DNA sequences and to intracellular degradation of the recombinant products by the host cell in which they are made. In addition, degradation of the recombinant products during purification process can also be problematic. For example, deamidation of asparaginyl (Asn) and glutaminyl (Gln) residues to produce aspartate (Asp) and glutamate (Glu) residues causes structurally and biologically important alterations in peptide and protein structures. Although deamidation occurs spontaneously in some proteins, the problem may be exacerbated in the expression and purification of recombinant proteins. (Robinson, N. E., et al., PNAS 98(22):12409-12413-208, 2001; Robinson, N. E., et al., PNAS 98(8): 4367-4372, 2001). Moreover, deamidation instability of recombinant produced proteins may be problematic in developing a preferred solution pharmaceutical formulation due to degradation of the recombinant protein after long-term storage.
The present invention solves the problem of deamidation associated with recombinant proteins by providing FGF-21 muteins that have a reduced amount of deamidation compared to wild type FGF-21. Applicants have found that the FGF-21 muteins with reduced deamidation can be produced in industrial fermentation conditions, can be pharmaceutically formulated in a solution form, and maintain the biological activity necessary to be useful to treat subjects with disorders including, but not limited to, type 2 diabetes, obesity, and metabolic syndrome.